IL-6 is a protein belonging to the group of cytokines, which proved to play a key role in the organism's immune response and haematopoiesis stimulation.
Many biological functions have, in fact, been found for IL-6 in the hematopoietic and lymphoid system, in the liver and in other target organs and cells. Some of these functions are beneficial, while others are related to pathological states. Among the latter functions, IL-6 has been found to be a growth factor for multiple myeloma cells; anti-IL-6 antibodies were shown to transiently block myeloma cell proliferation in a leukemic patient (see for example Klein et al., Blood, 78, (5), pp.1198-1204, 1991 and Lu et al., Eur. J. Immunol., 22, pp. 2819-24, 1992).
Elevated IL-6 levels have been correlated with autoimmune and inflammatory diseases, such as rheumatoid arthritis, glomerulonephritis, psoriasis, and Castelman's disease (see for example Graeve et al., Clin. Investig., 71, pp.664-71, 1993). IL-6 has also been shown to play a direct role in bone loss and hypercalcemia (see for example Poli et al., Embo J., 13, (5) pp. 1189-96 and Yoneda et al., Cancer Res., 53, pp. 737-40, February 1993).
The development of inhibitors of IL-6 activity has therefore been the subject of active research. For this purpose, different approaches have been pursued, including the use of antibodies against IL-6 (as reported by Klein et al. above), gp130 or gp80; the use of soluble gp130; or the use of muteins for IL-6, or IL-6 Receptor.
Since these approaches might be associated with specific unwanted effects in clinical applications (as reported by Lu et al., above), the setting-up of additional strategies to inhibit IL-6 activity would be useful.
The Applicant has, therefore, investigated a different approach to inhibit IL-6 activity: by blocking the intracellular proteins mediating the IL-6 signal.
The transduction of the IL-6 signal in responsive cells has been intensively investigated. Fowlkes et al. (PNAS USA, 81, pp. 2313-6, 1984) first suspected DNA responsive elements specific for IL-6 flanking the rat fibrinogen genes.
Later on, Kunz et al. (Nuc. Ac. Res., 17, (3), 1121-37, 1989) showed a responsive element with a core sequence identical to that of rat fibrinogen genes (CTGGGA) to respond to IL-6 in the rat .alpha..sub.2 -macroglobulin gene.
DNA responsive elements with sequences related to those above-mentioned have also been defined in the genes coding for the human C Reactive Protein (CRP) (see Toniatti et al., Mol. Biol. Med, 7, pp. 199-212, 1990), human haptoglobin (see Oliviero et al., Embo J. 6, (7), pp. 1905-12, 1987) and in other genes coding for additional acute phase proteins induced by IL-6 (see Heinrich et al., Biochem. J., 265, pp. 621-36, 1990), leading to the definition of a core consensus sequence CTGGGAW or CTGGRAA, where W stands for A or T, and R stands for A or G.
Hocke et al. (Mol. Cell. Biol., 12, (5), pp. 2282-94, 1992) indicated that multiple related core sequences, similar to the core sequence mentioned above, might be present in regulatory regions of wild-type genes responding to IL-6 and that this multiplicity leads to amplification of the response, as functionally analyzed with a reporter gene assay.
Wegenka et al. (Mol. Cell. Biol., 13, (1), pp. 276-88, 1993) have recently indicated an enlarged version of the core consensus sequence as the Acute Phase Response Element (APRE), active in hepatoma cells that can be represented by the formula KTMYKGKAA, wherein M stands for C or A, K stands for T or G, Y stands for C or T.
By Yuan et al. (Mol. Cell. Biol., 14, (3), pp. 1657-68, 1994) it has been shown that such APRE-like sequences bind a protein transcription factor having a molecular weight of about 90 KD, called APRF which has been recently cloned (see Akira et al., Cell, 77, pp. 63-71, 1994). In practice, the binding of activated APRF to APRE sequences would therefore lead to activation of IL-6-inducible genes (containing such APRE sequences) in IL-6-responsive cells.
As a consequence of this, an APRE element can be used as enhancer of a target gene in IL-6 responsive cells in the following way: in IL-6 responsive cells, the treatment with IL-6 induces the synthesis of APRF proteins, which bind to the APRE element, and such binding activates the expression of the target gene.
Serlupi Crescenzi et al. (Poster at the 12th European Imunol., Meeting, Barcelona, Jun. 14-17, 1994) have shown that an 8-fold repetition of the APRE DNA sequence (M8) is responsible for a 50-100 fold induction by IL-6 of a reporter gene in HepG2 human hepatoma cells.